The long-term objectives of this proposal are twofold: (1) to identify neurotransmitters released from rat and mouse taste bud cells during gustatory stimulation and (2) to investigate signal processing within the taste bud. These both are significant unanswered questions in the field of chemosensory reception. To date, there is only indirect evidence for candidate neurotransmitters in taste cells, including serotonin, glutamate, norepinephrine, acetylcholine, ATP, and peptides. We have developed a novel methodology to dentify the neurotransmitter(s) released from rat and mouse taste buds during gustatory stimulation. Our methodology uses Chinese Hamster Ovary (CHO) cells stably expressing high affinity receptors for candidate neurotransmitters and loaded with the calcium-sensitive dye, Fura 2. We will use these cells as biosensors to detect tastant-evoked release of neurotransmitters from isolated tasted buds and taste cells. The specific aims include investigating whether taste stimulation elicits release of serotonin, norepinephrine, acetylcholine, glutamate, ATP, CCK, and VIP from mourse and rat taste buds removed from vallate, foliate, palatal, and fungiform taste fields. Further, we aim to investigate whether such release is mediated by Ca2+ influx, intracellular Ca2+ release, or via transporter-mediated mechanisms. Finally, we propose to investigate cell-to-cell communication within taste buds via paracrine substances such as ATP and glutamate, and how information is transmitted from sensory receptor cells to synaptic output cells and on to primary sensory afferent terminals. The project will illuminate information processing in peripheral taste organs and may solve the conundrum of how signals generated in one cell (receptor cells) are transmitted to other cells (synaptic output cells) in the taste bud. The results may resolve the dilemma that taste cells appear to express only a specified set of taste receptors (i.e., bitter, or sweet, or umami GPCRs). Yet, some taste cells and many sensory afferent nerve fibers respond to multiple taste stimuli. The project will begin to raise our understanding of how taste signals are refined in the periphery, perhaps comparable to how visual signals are processed in the retina